Improved understanding of gene regulatory networks in stem and progenitor cells is essential for the development of strategies for manipulating progenitor cell fates for experimental and therapeutic purposes. MicroRNAs (miRNAs) are small non-coding regulatory RNAs that reduce translation and/or stability of complementary target messenger RNAs (mRNAs). miRNA represents a critical mechanism for the control of gene expression and cell fate specification in addition to transcription factors (TF) that are traditionally associated with this role. The hematopoietic system provides an outstanding model for investigation of the role of miRNAs in regulation of stem/progenitor cell fate since cell populations at different stages of differentiation are well characterized. Previous studies support a critical role for miRNA in regulating hematopoiesis but have only focused on a few candidate miRNAs. The relative abundance and specificity of expression for most miRNAs remains to be investigated, and the role of expressed miRNA singly or cooperatively in regulating gene expression and determining cell fate and function remains poorly understood. The goal of the proposed studies is to investigate the role of miRNAs in regulation of hematopoietic stem and progenitor cell differentiation and self-renewal. A multidisciplinary team of collaborators with expertise in hematopoiesis, RNA biology, bioengineering/nanotechnology and bioinformatics will develop and apply state- of-the-art technologies to address this important goal. Project 1 will study miRNA expression and gene regulation in hematopoietic stem and progenitor cells. miRNA expression and transcriptome composition in precisely defined progenitor populations will be studied using ultra-high throughput sequencing approaches and regulatory networks of miRNAs, transcription factors and their target genes at different stages of differentiation will be investigated. Project 2 will study the function of expressed miRNAs in lineage specification and self-renewal of hematopoietic stem and progenitor cells. High-throughput methods for screening miRNA function will be developed and putative function validated by detailed in vitro and in vivo analyses. Project 3 will study miRNA mediated gene regulation in hematopoietic stem and progenitor cells at the single cell level. A newly developed microfluidic bioprocessor will be used to investigate miRNA-mediated gene regulation in individual progenitor cells. The proposed studies have clear translational applications to manipulation of stem and progenitor cell populations for therapeutics, including transplantation, directed differentiation and gene therapy. These studies will also provide a basis for understanding perturbations in miRNA-mediated gene regulation in pathological states including malignancies. The sequencing, screening and bioengineering approaches developed for these studies have considerable potential for broader application within the consortium for investigation of the role of miRNA in cell fate determination in progenitor populations in cardiac, vascular and pulmonary tissues, and for investigating human tissue specific stem cell development from pluripotent stem cells.